It is often desirable to investigate small objects, such as biological tissues and chemical molecules. To that end, photonic excitation has application in both microscopy and spectroscopy. Broadly, the target object interacts with incident photons and emits a corresponding photon which is detected and used to generate an image of the target object.
Two-photon excitation microscopy involves the simultaneous absorption by a fluorophore of two photons of relatively low energy, causing emission by the fluorophore of a single fluorescence photon. More specifically, an intrinsic fluorophore or a fluorescent dye attached to the target is excited by the incident photons and emits the fluorescent photon which can be used to produce an image of the target. This technology allows for deeper penetration and higher resolution than conventional confocal microscopy.
The probability of simultaneous absorption of two photons is relatively low but increases quadratically with excitation intensity. As such, a strongly focused, subpicosecond pulse laser is typically used as the source of excitation photons. Scanning the laser beam allows for collecting two-photon excited fluorescence from multiple points on the target, from which a comprehensive image of the target can be constructed. The highly localized character of two-photon excitation and the use of near-infrared wavelengths minimize damage to the target and reduce the autofluorescent background noise experienced in confocal microscopy. Unfortunately, applications for two-photon microscopy have been limited by the requirement of using a relatively large and expensive solid state laser, such as a Ti:Sapphire laser, as the source of excitation photons. Furthermore, wavelength switching with a solid state laser requires mechanical realignment of the laser and can be difficult to accomplish.
Coherent Raman imaging and spectroscopy exploits the Raman effect in which incident light is scattered at a wavelength shifted by the energy of a molecular vibration, either to lower energy (longer wavelength) in Stokes Raman scattering or to higher energy (shorter wavelength) in anti-Stokes Raman scattering.
Coherent Anti-Stokes Raman Scattering (CARS) involves the nonlinear conversion of two laser beams into a coherent Raman beam of high intensity in the anti-Stokes region. The resulting emission is stronger than normal Raman scattering because of coherent interaction of light with the sample. This technique allows for obtaining high-quality three-dimensional images. CARS does not require fluorescent labeling of the target object; instead, different molecules are identified by the strengths and wavelengths of the emitted Anti-Stokes light. Unfortunately, CARS imaging and spectroscopy require the use of two different and expensive laser sources, such as two Ti:Sapphire lasers, which must be stringently synchronized and maintained. Such synchronization is particularly difficult because the widths of the optical pulses are on the order of 100 femtoseconds, i.e., 100×10−15 seconds.